This application addresses broad Challenge Area (06): Enabling Technologies, and specific Challenge Topic, 01-OD-101: Development of new tools and technologies to interrogate human mitochondrial function in vivo. Dysfunction of Mitochondria (MT) dysfunction is thought to be a primary contributor to aging and neurodegenerative disease. Although MT function is measurable by well-established properties of oxidative phosphorylation and ATP production, these defined measures have not directly led to an obvious understanding of pathophysiology. Alzheimer's (AD) Parkinson's (PD) Huntington's disease shares the property that MT is not keeping up with the energy demands of the cell. However, AD, PD, and HD display region-specific cell death, and even within those regions, only select cell types are targeted. Regional specificity, therefore, implies that MT dysfunction develops in response to the changing cellular metabolism in only specific cells with age or as disease progresses. Due to system heterogeneity, there have been substantial barriers that have made it difficult to determine whether the primary defect is the inability of particular MT to convert metabolic precursors to ATP, or a failure of cellular pathways to generate sufficient metabolic precursors for ATP synthesis. To overcome these limitations, we have developed a completely new, ultra-high sensitivity mass spectrometry technique called Nanostructure-Initiator Mass Spectrometry (NIMS), which is capable of mass analysis at 150nm resolution. Thus, NIMS has the unprecedented ability to spatially track mass composition in a single cell or in a single MT. NIMS is designed to create molecule signature that uniquely distinguish among even adjacent cell types, and does not destroy the 3-dimenational architecture of the brain. We have adapted NIMS to image and localize individual cells by their metabolite composition in any region within whole brain slices, and to directly measure MT-specific variations in single cells in situ. Issues with fragmentation, sensitivity, and low spatial resolution have limited the use of other mass spectroscopy techniques to achieve the resolution and imaging capabilities NIMS. However, our approach provides, perhaps the only existing technological advance that efficiently and precisely resolve, region, cell type, and MT-specific heterogeneity. NIMS can be applied to any disease, to any tissue section, and is idea to sort out complex changes that occur in the brain. PUBLIC HEALTH RELEVANCE: At least 2% of Americans will be afflicted with some form of Alzheimer's disease (AD) (4,000,000), Parkinson's disease (PD) (1,500,000), or Huntington's disease (HD) (200,000), among others. Each of these disorders can affect patients for decades, yet no effective long-term approaches to therapy are currently available. Because the number of affected individuals will grow exponentially, the gap between the problem's size and our capabilities for treatment will widen. Thus, an understanding of the pivotal role of MT dysfunction promises to increase therapeutic options.